1. Field
This disclosure is concerned generally with purified immunoglobulins and specifically with a highly purified immunoglobulin of the IgM class which is substantially free of nucleic acids.
2. Prior Art
IgM is a well known 19S immunoglobulin which comprises about 7% of the immunoglobulins found in man. IgM antibodies are said to have an antibody valence of at least five and they are the earliest antibodies generated in an immune response. Although IgM antibodies tend to be very effective, especially in combating bacterial infections, they have a relatively short in vivo half life of about five days. Further, IgM antibodies are labile and relatively difficult to stabilize, especially in purified form.
Various purification schemes have been suggested for plasma-derived IgM and, more recently, monoclonal-derived IgM. In the case of plasma-derived IgM, it has been known since the 1940's that alcohol fractionation techniques could be used to obtain a relatively concentrated IgM from what is known as Rohn Fraction . See for example U.S. Pat. No. 4,318,902 (and the cited references) to W. Stephan and concerned with the use of beta-propriolactone to make a concentrated IgM suitable for intravenous (IV) administration. In addition, see EPO application 0 038 667 of Miura et al (IgM acylation). See also, U.S. Pat. No. 4,272,521 to Zuffi concerned with the purification of immune serum globulins in general by using ion exchange resins at an alkaline pH. Other IgM purification or preparation techniques are disclosed by U. Sugg et al, Vox Sang. 36:25-28 (1979); M. Steinbach et al, Preparative Biochemistry 3(4), 363-373 (1973) and A. Wichman et al, Biochem. Biophys. Acta 490:363 -69 (1977). Techniques for making specific monoclonal antibodies of the IgM type are shown in U.S. Pat. No. 4,271,145 to Wands et al. A specific immunoassay using high affinity IgM antibodies is disclosed in W 082/01072 published in the names of Wands et al. See also, I. A. Sampson et al, J. Immuno. Meth. 69, pp. 9-15, 1984. For a variety of technical reasons, plasma derived IgM has been relatively difficult to purify and the highest known purity to dare is about 90% IgM, by weight. Also, the nucleic acid content of such plasma derived IgM has generally not been a serious concern because the IgM is derived from a human plasma source.
Typical nucleic acid contents for plasma-derived IgM are thought to be in the range of about 1 ng to 10 .mu.g per mg.
Since the publication by Kohler and Milstein, "Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity", Nature 256:495-497 (1975), the production of monoclonal antibodies has become well known. Monoclonal antibodies of a given specificity are now routinely made using somatic cell hybrids (see, for example, U.S. Pat. No. 4,172,124 to H. Koprowski et al), using EBV transformed cells (see, for example, U.S. Pat. No. 4,446,465 to M. Lostrom), a combination of the two or by the electrofusion of cells. Monoclonals of both the IgG and IgM classes have been made, purified and characterized. Such IgM preparations are described by D. Nau, Biochromatography, 1, No. 2, pp. 83-84 (95% pure IgM from tissue culture); M. Fishner, U.S. Pat. No. 4,604,235 (90% pure IgM from mouse asciter fluid and which was characterized as "essentially pure antibody"); J. R. Wands et al, W 082/01072 (high affinity IgM monoclonal antibodies for diagnostics, cited above); S. Burchiel, et al, J. Immuno. Meth., 69, p. 33, 1984 (IgG purified from mouse asciter fluid); J. Deschamps et al, Anal. Biochem. 147, p. 451, 1985 (IgG from mouse asciter fluid); and T. Brooks et al, Amer. Lab., October, 1985 (use of hydroxyapatite for purification of mouse and human IgG and IgM). Although efforts have been made to purify IgM obtained from monoclonal sources, the highest reported IgM purity to date is about 95% (see Nau, above).
The preparation of a monoclonal IgM against P. aeruginosa has been disclosed and IgM derived from a human lymphoblastoid tissue culture and DEAE Sephacel has been used for an initial purification of the IgM. Therapeutically acceptable isotonic solutions of IgM with a concentration of 0.005 to 0.5 ug/ml are known but no data has been given on the relative purity of the IgM product or its formulation.
Although nucleic acid content of plasma-derived IgM has not aroused significant concern, the nucleic acid content of monoclonal IgM is very significant because of the potential danger of introducing foreign (non-human) nucleic acid into a human via a parenterally administered product. Hence, in addition to the desirability of obtaining a purified and concentrated IgM product, it is also desirable to obtain such a product with no or very little nucleic acid. We have now found that such a purified product can be prepared and stabilized by carefully controlling the processing steps and storage conditions. Details of our highly purified IgM are described below.